Protein modification by small ubiquitin-like modifiers (SUMOs) regulates the activity and fate of a subset of transcription and signaling proteins, and often their subcellular location. Sumoylation is often a pre-requisite for ubiquitination, and therefore influences the ultimate fate of ubiquitinated proteins. Consequently, the regulation of sumoylation is considered important in the ultimate disposal or aggregation of subsets of cellular proteins implicated in neurodegeneration. Protein sumoylation is regulated by two opposing reactions: conjugation - which is carried out by a system of enzymes that activate SUMO and couple it to targets, and deconjugation - which is carried out by members of a specialized protease family. This proposal seeks to generate unique tools to study and track the activity of the seven known human Senps (sentrin specific proteases - sentrin being a previous name for SUMO). These enzymes are thought to be primarily involved in both processing of immature SUMO (endopeptidase activity) and removal of SUMO after conjugation to target proteins (isopeptidase activity). A chemistry based approach will be utilized to develop small-molecule probes that irreversibly inactivate individual Senps by reaction with critical active site residue of enzyme. The activity based probes are expected to be useful to control the activity of proteases and help determine the physiological consequences of their proteolytic activity. Tagging probes with biotin will facilitate the identification of target enzymes in tissue lysates, intact cells, and even potentially in vivo. We anticipate obtaining potent and selective Senp inhibitors and probes that have a great potential to become widely use technology in exploring the biology and pathology of sumoylation. PUBLIC HEALTH RELEVANCE: Protein modification by small ubiquitin-like modifiers (SUMOs) regulates the activity and location of an expanding array of cellular proteins, and is implicated in the onset of neurodegenerative disease. Protein sumoylation is regulated by two opposing reactions: conjugation - which is carried out by a system of enzymes that activate SUMO and couple it to targets, and deconjugation - which is carried out by members of a specialized protease family. This proposal seeks to generate unique tools to study and track the activity of the specific proteases involved in deconjugation, and is a crucial step in determining exactly how SUMOs are involved in disease.